The overall objective of this proposal is to develop improved methods for somatic gene therapy for treatment of lung disease in cystic fibrosis (CF). The rationale is that there is a great need for improved delivery systems which will combine high efficiency for transfection, delivery to the proper airway cells in the lung, and longer duration or permanent expression. All of the projects and almost all of the pilot projects are focused on this general objective. Project l has the following aims: reduction of toxicity of adenovirus (Ad) vectors using temperature sensitive and other mutations; development of Ad vectors based on alternative serotypes and nonhuman viruses; and development of Ad vectors for persistent expression. Project 2 has the following specific aims: development of Ad vectors accommodating much larger inserts (one use of which would be to include large segments of DNA to promote homologous recombination and permanent expression); and development of Ad vectors incorporating a regulatable promoter. Project 3 has the following specific aims: development of chemically defined delivery systems based on DNA conjugates using synthetic components to achieve receptor/Iigand binding, lysis of endosome, and nuclear targeting; and preparation of DNA conjugates for targeting to airway epithelium using ligands for the receptors for folate, mannose 6-phosphate, vasoactive intestinal peptide, and dimeric IgA. Project 4 has the following specific aims: comparison of aerosolization, tracheal instillation, and bronchial artery injection for delivery of Ad vectors to the airway epithelium; gathering .preclinical data for use of these routes of administration in humans; development of a program for clinical trials beginning with assessment of the natural history of Ad infections in CF patients and clinical electrophysiological measurements; assessment of bronchial artery and aerosol delivery of vectors developed in projects l and 3 and of AAV vectors from collaborators; and efficacy studies using CF mutant mice. Core A will prepare Ad vectors and retroviral vectors to meet FDA criteria for preclinical studies and human trials. Core B will provide an outstanding capacity for primate studies using new vectors and delivery systems from all four projects. Core C will provide histopathology, immunohistochemistry, and in situ hybridization studies. Topics of pilot projects include use of gene therapy for prevention of coronary restenosis, gene therapy for intestinal involvement in CF, development of additional mutations in the CF gene in the mouse, use of retroviral vectors for airway delivery, and development of a retroviral vector for nondividing cells.